CN117866751A - Method and apparatus for DMF-based digital nucleic acid amplification systems - Google Patents

Method and apparatus for DMF-based digital nucleic acid amplification systems Download PDF

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CN117866751A
CN117866751A CN202311645951.XA CN202311645951A CN117866751A CN 117866751 A CN117866751 A CN 117866751A CN 202311645951 A CN202311645951 A CN 202311645951A CN 117866751 A CN117866751 A CN 117866751A
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bottom plate
water bath
unit body
hydrophilic
hot water
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孟李
李明忠
吕阿曼
贾艳伟
麦沛然
马许愿
罗文基
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University of Macau
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University of Macau
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Abstract

The invention belongs to the technical field of digital microfluidics, and particularly relates to a method and a device for a digital nucleic acid amplification system based on DMF. The invention provides a bottom plate with a hydrophobic-hydrophilic array, which is used for generating a large number of uniform micro-droplets on a DMF chip, so that accurate absolute quantification of nucleic acid molecules is realized. The substrate is rendered hydrophobic-hydrophilic by a hot water bath treatment, silane modification and uv ozone partial exposure. When the size of a single hydrophilic pattern is 200 mu m, the invention realizes passive separation of lambda DNA sample with dynamic range of 1-1000 copies/mu l into 1818 independent micro-droplet reaction areas, the 3.4% volume difference accords with poisson distribution requirement of digital nucleic acid amplification, and the droplets automatically move and have accurate reaction volume. In addition, the digital loop-mediated isothermal amplification performed on the digital microfluidic device has accurate result and small error, and is superior to the relative quantitative nucleic acid amplification of the traditional PCR equipment.

Description

Method and apparatus for DMF-based digital nucleic acid amplification systems
Technical Field
The invention belongs to the technical field of digital microfluidics, and particularly relates to a method and a device for a digital nucleic acid amplification system based on DMF.
Background
Quantification of nucleic acid molecules plays an important role in clinical diagnosis, pathogenic microbiological examination and genomic research. Real-time Polymerase Chain Reaction (PCR) or isothermal amplification can be relatively quantified by comparison to standard curves of known concentration. However, the dependence on the amplification efficiency of different reactions may significantly affect the accuracy of the results. Digital nucleic acid analysis (Digital nucleic acid analysis, dNAA) is a new generation of absolute quantitative analysis technology, gets rid of dependence on standard curves, and has higher sensitivity and accuracy. Dividing the nucleic acid sample into a large number of equal volume partitions is a key and necessary step for poisson statistical analysis of dNAA. The channel microfluidic based approach can create the large number of microreaction partitions required by dNAA, but the complex external valves, pumps and channels in the channel microfluidics can limit the flexibility of the system. In addition, it presents a potential risk of tubing blockage and formation of dead volumes, which would affect the accuracy of dNAA results.
As an emerging liquid handling technology, digital microfluidic (Digital microfluidics, DMF) can simultaneously manipulate discrete droplets based on electrowetting on medium (Electrowetting on dielectric, EWOD) effects, enabling the dispensing, movement, merging and splitting of droplet volumes from nanoliters to microliters. However, due to the inherent limitations of the EWOD effect, the droplet size dispensed by conventional DMF systems is limited by the electrode size. Furthermore, conventional digital microfluidic devices typically use a direct addressing scheme to control the electrodes, where each electrode is individually activated by a dedicated control pin connected to a signal line. Although this approach may achieve the greatestControl flexibility, but for practical digital microfluidic devices it requires too many control pins and the crowded arrangement of signal lines between electrodes can affect droplet movement. This results in a DMF electrode count of difficult to exceed 200 and further limits the throughput and number of droplets that can be generated. Furthermore, common droplet separation schemes require three electrode fits, lack flexibility, and large variations in volume between different droplets. Functional TiO despite super wettability contrast 2 The substrate can implement a series of microdroplets (CN 116068014 a) on a DMF platform, but the manufacturing process is toxic, cumbersome, time-consuming and energy-consuming. These problems have so far prevented the push of DMF based dNAA. Therefore, development of a DMF platform capable of generating a large amount of uniform droplets in a low-cost and easy-to-manufacture manner is imperative, and the DMF platform will be suitable for high-throughput applications such as digital loop-mediated isothermal amplification (dLAMP).
Disclosure of Invention
An object of a first aspect of the invention is to provide a base plate.
The object of the second aspect of the present invention is to provide a method for manufacturing a base plate according to the first aspect of the present invention.
The third aspect of the present invention is directed to a microfluidic chip.
The fourth aspect of the present invention is directed to a detection method.
A fifth aspect of the present invention is directed to a detection system.
In order to achieve the above purpose of the present invention, the present invention adopts the following technical scheme:
in a first aspect of the invention, there is provided a base plate:
the bottom plate comprises a hydrophilic porous micro-nano structure surface and a hydrophobic film.
Preferably, the hydrophobic film does not completely cover the hydrophilic porous micro-nanostructure surface.
Preferably, the hydrophilic porous micro-nano structure surface is formed with a hydrophilic pattern at a place not covered by the hydrophobic film.
Preferably, the hydrophilic pattern is composed of a plurality of circular or rectangular unit cell arrays.
Preferably, the individual diameter or side length of the unit cell is 50-500 μm.
Preferably, the diameter or side length of the unit cell is 150-250 μm.
Preferably, the gap between the unit body and the adjacent unit body is 50-500 μm.
Preferably, the gap between the unit body and the adjacent unit body is 150-250 μm.
Preferably, the contact angle of the hydrophilic porous micro-nanostructure surface is less than or equal to 10 °.
Preferably, the contact angle of the hydrophobic film is greater than or equal to 150 °.
Preferably, the material of the bottom plate comprises one of beryllium, magnesium, aluminum, scandium, titanium, vanadium, chromium, iron, manganese, cobalt, nickel, copper, zinc, molybdenum, cadmium, indium, tin, antimony, bismuth, tantalum, thallium, lead, neodymium and erbium or an alloy composed of the same.
Preferably, the material of the bottom plate comprises one of magnesium, aluminum, zinc, iron, lead and manganese or an alloy composed of the magnesium, the aluminum, the zinc, the iron, the lead and the manganese.
Preferably, the material of the bottom plate comprises one of aluminum and magnesium or an alloy consisting of the aluminum and the magnesium.
Preferably, the bottom plate is made of aluminum.
Preferably, the hydrophobic film is an organosilane.
Preferably, the organosilane includes at least one of chlorotrimethylsilane, monochloro-propyl dimethyl silane, octyltrimethoxysilane, dodecafluoroheptyl propyl trimethoxysilane, tridecafluorooctyl triethoxysilane, trichloro (1H, 2H-perfluorooctyl) silane, perfluorodecyl triethoxysilane, heptadecafluorodecyl trimethoxysilane, and heptadecafluorodecyl triethoxysilane.
Preferably, the organosilane is perfluorodecyl triethoxysilane.
Preferably, the hydrophobic film is a monolayer.
In a second aspect of the present invention, there is provided a method of manufacturing a base plate of the first aspect of the present invention, comprising the steps of:
1) Forming a hydrophilic porous micro-nano structure surface on the bottom plate by adopting a water bath method;
2) Depositing a hydrophobic film on the bottom plate by adopting a vapor deposition method;
3) Ultraviolet rays are adopted to decompose the hydrophobic film in a specific area, so that a hydrophilic pattern is formed.
Preferably, in the step 1), a water bath method is adopted to form a hydrophilic porous micro-nano structure surface on the bottom plate, and the method specifically comprises the following steps: the bottom plate is placed in a hot water bath for treatment.
Preferably, the vapor deposition method in step 2) is a chemical vapor deposition method, specifically: and depositing a hydrophobic material on the surface of the porous micro-nano structure to form a hydrophobic film.
Preferably, in the step 3), the hydrophobic film in the specific area is decomposed by ultraviolet rays to form a hydrophilic pattern, specifically: the photomask with specific pattern is used for shielding the bottom plate, and ultraviolet rays penetrate through the photomask pattern to decompose the hydrophobic film in the specific area, so that a hydrophilic pattern is formed.
Preferably, the hot water bath treatment time is 0-24 hours.
Preferably, the hot water bath treatment time is 2-60min.
Preferably, the hot water bath treatment time is 5-20min.
Preferably, the hot water bath treatment temperature is 50-100 ℃.
Preferably, the hot water bath treatment temperature is 60-90 ℃.
Preferably, the hot water bath treatment temperature is 70-80 ℃.
Preferably, the time of deposition is 10-60min.
Preferably, the time of deposition is 25-35min.
Preferably, the temperature of the deposition is 150-250 ℃.
Preferably, the temperature of the deposition is 180-220 ℃.
Preferably, the ultraviolet rays are dual wavelength ultraviolet rays.
Preferably, the wavelength of the ultraviolet rays is 180-190nm and 250-260nm.
Preferably, the ultraviolet irradiation time is 15-60min.
Preferably, the photomask has a unit body that is transparent to ultraviolet rays.
Preferably, the unit body is circular or rectangular with a diameter or side length of 50-500 μm.
Preferably, the diameter or side length of the unit cell is 150-250 μm.
Preferably, the gap between the unit body and the adjacent unit body is 50-500 μm.
Preferably, the gap between the unit body and the adjacent unit body is 150-250 μm.
In a third aspect, the present invention provides a microfluidic chip, which is characterized by comprising a middle spacer, a driving top plate and a bottom plate according to the first aspect of the present invention.
Preferably, the bottom plate is positioned at the bottom, the middle gasket is positioned in the middle, and the driving top plate is positioned above, so that a three-layer structure is formed.
Preferably, the material of the middle pad includes at least one of glass, ceramic, metal, and dimethyl siloxane.
Preferably, the height of the intermediate pad is 200-500 μm.
Preferably, the driving top plate is a glass plate covered with a transparent conductive layer.
Preferably, the thickness of the driving top plate is 1.1mm.
Preferably, the transparent conductive layer has a thickness of 30-300nm.
Preferably, the transparent conductive layer includes at least one of indium tin oxide ITO, aluminum doped zinc oxide AZO, and fluorine doped tin oxide FTO.
Preferably, a side of the driving top plate facing the bottom plate is provided with a driving circuit.
Preferably, the driving circuit is formed of a transparent conductive layer.
Preferably, the driving circuit forms 4-8 channels.
Preferably, there are 12-24 electrodes per channel.
Preferably, the electrode area comprises (2.2-2.8 mm) × (2.2-2.8 mm).
Preferably, the electrode adjacent gap comprises 50-70 μm.
Preferably, the side of the driving top plate facing the bottom plate is provided with an insulating coating.
Preferably, the insulating coating surface has a hydrophobic coating.
Preferably, the insulating coating comprises at least one of parylene or ultraviolet curable glue.
Preferably, the ultraviolet curing glue comprises resin, photoinitiator and auxiliary agent.
Preferably, the resin comprises at least one of phenolic resin, ABS resin, polyvinyl chloride resin, rosin, and amber.
Preferably, the photoinitiator includes at least one of benzoin ethers, phthalide ketals, acetophenones, benzophenones, thioarrowheads, aromatic sulfonium salts, iodonium salts, and ferrocenium salts.
Preferably, the auxiliary agent comprises at least one of an industrially acceptable solvent, a stabilizer, a leveling agent, an antifoaming agent, a plasticizer and a coupling agent.
Preferably, the hydrophobic coating comprises teflon.
In a fourth aspect of the present invention, there is provided a detection method, wherein an oil phase liquid is added to the microfluidic chip according to the third aspect of the present invention, and then a detection sample is added to perform driving separation and detection.
Preferably, the oil phase liquid comprises at least one of n-hexadecane, silicone oil and mineral oil.
Preferably, the method is used for quantitative detection of nucleic acids.
Preferably, the nucleic acid quantitative detection can be applied to at least one of a genetic mutation detection virus detection, a copy number variation detection, and a transgenic food detection.
Preferably, the test sample comprises deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
Preferably, the test sample is lambda DNA.
An object of a fifth aspect of the present invention is to provide a detection system comprising a microfluidic chip according to the fourth aspect of the present invention, the system further comprising any one or more of:
1) A temperature control system including one or more of a heating system, a cooling system, a temperature sensing element, a temperature display system, a temperature control panel, and the like.
2) The sample injection and sample outlet system comprises one or more of a sample inlet, a sample outlet, a cleaning device, a drying device and the like.
3) A data processing system including one or more of a signal collection system, a computer processing device, a storage device, an operating system, and the like.
4) The result display system, which may also be referred to as a result transmitting device, may be specifically one or more of a Liquid Crystal Display (LCD), a Light Emitting Diode (LED) display, a plasma display, a projection display, a touch screen display, and the like, and the result transmitting device may transmit the detection result of the sample to an information communication terminal device that the user may refer to.
The beneficial effects of the invention are as follows:
the invention proposes a substrate with surface micro-nanostructures and hydrophilic-hydrophobic phase-separated regions, integrated with a DMF system, for absolute quantification of nucleic acids. The combination of super-wettability contrast and EWOD effects can successfully produce a droplet array. Super hydrophilic dots with a certain size and high wettability contrast can ensure a uniform droplet volume. In addition, the separation in the oil phase can effectively avoid the problems of liquid evaporation and cross contamination, and the DMF structure without a pipeline and a valve can avoid the formation of dead volume. The proposed DMF platform can successfully generate 1818 highly uniform droplets while successfully achieving absolute quantification of lambda DNA in 60 minutes with a dynamic range of 1 to 1000 copies/microliter.
Drawings
FIG. 1 is the effect of different treatment conditions on the surface structure of an aluminum sheet, wherein A represents the untreated aluminum sheet surface; b represents the surface of the aluminum plate treated at 55 ℃ for 10 minutes; c represents the surface of the aluminum plate treated at 75 ℃ for 10 minutes; d represents the surface of an aluminum plate treated at 85℃for 10 minutes.
FIG. 2 is an effect of different process conditions on the Contact Angle (CA) and Slip Angle (SA) of a substrate, wherein A is the contact angle change of the substrate after hot water treatment at different temperatures for different times, without CVD deposition; b is the contact angle change of the bottom plate after the CVD deposition in different times of hot water treatment at different temperatures; c is the sliding angle change of the bottom plate after the CVD deposition in different times of hot water treatment at different temperatures; d is the contact angle change of the bottom plate under different ultraviolet irradiation time after the bottom plate is treated by hot water at different temperatures for different times and is deposited by CVD.
Fig. 3 is a schematic diagram of digital microfluidic chip preparation and structure: wherein A is a schematic diagram of top plate preparation and structure; b is a schematic diagram of the preparation and structure of the bottom plate; c is a schematic structural diagram of the assembled digital micro-fluidic chip.
FIG. 4 is a graph of the effect of superhydrophilic pattern size on uniformity of droplets produced: wherein A is a DMF chip cross-sectional view; b is a schematic diagram of the size of 1818 super-hydrophilic patterns; c is a 3D surface map of different sized droplets on the superhydrophilic pattern (150 μm, 200 μm, and 250 μm); d is a graph of the optical path difference between droplets.
Fig. 5 is a graph of experimental results of accuracy of reaction volumes of digital microfluidic chips: wherein A is a schematic diagram of droplet separation; b is a top-down fluorescence image of the liquid drops with different volumes; c is the result of the linear relationship between the volume of the liquid drop and the area thereof; d is a top view of the liquid volume after passing 5 μl of liquid droplets through 6 channels in sequence; e is the volume change result of 5 mu L liquid drops after passing through 6 channels in sequence; f is the result of the volume of liquid left on each channel after 5 μl droplets pass through 6 channels in sequence.
Fig. 6 is a diagram of the results of a real-time imaging of the digital microfluidic detection system.
FIG. 7 is a schematic diagram of LAMP detection nucleic acid quantification using a digital microfluidic detection system.
FIG. 8 shows the LAMP reaction results at different reaction times.
FIG. 9 shows the LAMP reaction results at 60 min: wherein A represents the change result of the concentration of the DNA template and the number of fluorescent spots; b represents the linear relation between the concentration of the DNA template and the change of the quantity of fluorescent spots; c represents the measurement results of DMF-dLAMP and common commercial PCR instrument-qLAMP (Bio-Rad) on unknown concentration liquid.
FIG. 10 shows the detection results and standard curves of a standard sample by a common commercial qPCR instrument (Bio-Rad).
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
Examples part chemical and reagent sources are:
NOA68 uv curable glue was purchased from Norland Products inc (united states). Teflon-AF 1601S was purchased from DuPont, U.S.A. 1H, 2H-perfluorodecyl triethoxysilane and hydroxynaphthol blue (HNB) are available from Aba Ding Shenghua technologies Co., ltd (China). N-hexadecane was purchased from Sigma Aldrich (united states). Lambda DNA was purchased from Takara Bio Inc. (Japan). Evagreen was purchased from Biotium (USA). dNTPs are purchased from Applied Biosystems (UK). Bst 2.0 Hot Start DNA polymerase, mgSO 4 And 10 XThermolpol buffer (200 mM tris-HCl, 100mM (NH) 4 ) 2 SO 4 、500Mm KCl、20mM MgSO 4 1% Tween 20) was purchased from New England Biolabs inc (china). All chemicals and reagents were used as received without further purification. Deionized water used in all experiments was purified using a Millipore system (18.0 mΩ cm, milli-Q gradient system).
Indium Tin Oxide (ITO) coated glass (< 17ohm/sq,100 x 1.1 mm) and conventional glass (100 x 0.4 mm) were purchased from loma glaucor glass ltd (china). Aluminum plates (99.5%, 63×63×1 mm) were purchased from Shenzhen spring metal company (china). The photomask plate is designed by AutoCAD and purchased from Shenzhen micro-nano trade company (China).
The invention designs a hairpin fluorescent Dye (Reference Dye-FAM) similar to a molecular beacon. It consists of a probe sequence (loop) and complementary 3 'and 5' ends (stems), both of which carry the fluorophore FAM. The sequence is as follows:
5'6-FAM-GCCGCTTGACTTTCATTGGCTAATGCTAAATCCCAAGCGGC-3'6-FAM(SEQ ID NO.1)。
the specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1 influence of different hot bath treatment conditions on micro-nano structures on aluminum plate surface
The present example tested the effect of different treatment conditions on the micro-nano structure of the aluminum plate surface.
The surface was sanded with an aluminum plate having a purity of 99.5% and a size of 63.5mm×63.5mm and a thickness of 1mm, and the oxide film was removed. Control group (without hot water bath treatment), experimental group 1 (treatment temperature 55 ℃ C., treatment time 10 min), experimental group 2 (treatment temperature 75 ℃ C., treatment time 10 min), experimental group 3 (treatment temperature 85 ℃ C., treatment time 10 min) were set, and the surface structures of the aluminum plates under different treatment conditions were observed by using a scanning electron microscope, and the results are shown in FIG. 1.
The results show that the initial smooth surface of untreated aluminum panels was transformed into different micro-nanostructures after hot water bath treatment, and that a in fig. 1 represents the surface of aluminum panels of experimental group 1 that were not subjected to hot water bath treatment; in FIG. 1, B shows the surface of an aluminum plate of experiment group 2 (treated with a hot water bath at 55 ℃ C. For 10 minutes), at which time nano-grass-like bayerite (. Beta. -Al) was observed 2 O 3 ·3H 2 The micro-nano structure of O); in FIG. 1C, the surface of the aluminum plate in experiment group 2 (treated by a hot water bath at 75 ℃ for 10 minutes), obvious flower-like micro-nano structures can be observed, and the roughness is higher; in FIG. 1, D shows the surface of an aluminum plate of experiment group 3 (treated at 85 ℃ C. For 10 minutes), it can be seen that the surface micro-nano structure size is reduced, resulting in the formation of a loose and fragile boehmite film (α -Al 2 O 3 ·H 2 O)。
EXAMPLE 2 Effect of different Hot bath treatment conditions on floor wettability
This example tests the wettability effect of the surface of a base plate produced by different hot water bath treatment conditions.
The surface was sanded with an aluminum plate having a purity of 99.5% and a size of 63.5mm×63.5mm and a thickness of 1mm, and the oxide film was removed. Selecting time parameters of 0min, 5min, 10min, 15min and 20min; the temperature parameters are 55 ℃, 65 ℃, 75 ℃ and 85 ℃ and the combination design experiment is carried out to obtain 20 hot water bath treatment conditions. After the hot water bath treatment, thoroughly flushing with deionized water and blow-drying with nitrogen; treating the soleplate with oxygen plasma for 2 minutes; the plates were covered with a petri dish and 10 microliters of 1h,2 h-perfluorodecyl triethoxysilane were edge-dropped onto the plates and the plates were heated to 200 degrees for 30 minutes.
Contact Angle (CA) and Slip Angle (SA) were measured using 10. Mu.L of water droplets, and the results are shown in FIG. 2 as A-D, wherein A was not subjected to CVD deposition, B-C was subjected to CVD deposition, D was obtained by subjecting the resulting film to ultraviolet irradiation (185 nm,254nm,30 mW/cm) 2 ) The result of the hydrophilicity recovery. None of a-D uses photomasks to form a specific hydrophilic pattern.
In FIG. 2A, the CA measured without CVD deposition treatment when the aluminum substrate was subjected to hot water at 75℃or more for 10 minutes or more and hot water at 85℃or more for 5 minutes or more is less than 10 DEG, indicating that the aluminum substrate was super-hydrophilic at this time.
In FIG. 2, B shows that after CVD deposition treatment, the CA measured after the aluminum substrate is subjected to hot water at 65 ℃ or higher for 5 minutes is greater than 150 degrees, indicating that the aluminum substrate after the hydrophobic material is deposited has superhydrophobicity.
In fig. 2C shows the relationship between the slip angle of the substrate and the reaction temperature and time after the CVD deposition process, the substrate treated at 55℃ showed no SA, while the other three temperatures were observed. When the substrate is treated at 75℃for 10-15 minutes or 85℃for 5-10 minutes, its SA is 0, indicating that its hydrophobic properties are optimal. Based on CA and SA measurements, a hot water bath treatment at 75deg.C for 10 minutes was selected for the floor preparation and subsequent experiments.
In FIG. 2D, the hydrophilicity was measured after direct ultraviolet irradiation without using a photomask, and the change in the substrate after deposition of 1H, 2H-perfluorodecyl triethoxysilane was observed with the irradiation time of ultraviolet rays/ozone. It can be observed that the substrate surface became super hydrophilic after 10 minutes of irradiation and CA decreased to 0 ° after more than 15 minutes of irradiation, indicating that the hydrophobic layer can be decomposed by uv/ozone and a specific hydrophilic pattern can be formed by custom photomask.
Example 3A substrate having a porous micro-nanostructured surface with 2070 hydrophilic patterns
By way of examples 1-2, the treatment conditions for the hot water bath were determined to be 75℃for 10min.
An aluminum plate (purity: 99.5%, size: 63.5 mm. Times.63.5 mm, 1mm thick) was selected as a base plate material, and a base plate having a porous micro-nano structured surface with 2070 hydrophilic patterns was prepared as follows.
The experimental procedure was as follows:
1) Polishing the surface of the aluminum plate by sand paper to remove the oxide film;
2) The aluminum plate is treated by hot water bath, the hot water bath treatment condition is selected to be 75 ℃, and the time is 10min;
3) Thoroughly flushing with deionized water and blow-drying with nitrogen;
4) Treating the soleplate with oxygen plasma for 2 minutes;
5) Chemical vapor deposition: the plate was covered reversely with a petri dish and 10 μl of 1h,2 h-perfluorodecyl triethoxysilane was dropped onto the edge, and the plate was heated to 200 degrees for 30 minutes;
6) A photomask shielding base plate having a square void cell with a side length of 150 μm and a gap of 200 μm between each cell was used, and an ultraviolet ozone cleaner (185 nm,254nm,30mW/cm was used 2 ) Irradiating for 20min, and decomposing the hydrophobic film in the specific area through the photomask pattern to form 2070 hydrophilic patterns.
Example 4A substrate with a porous micro-nanostructure surface having 1818 hydrophilic patterns
An aluminum plate (purity: 99.5%, size: 63.5 mm. Times.63.5 mm, 1mm thick) was selected as a base plate material, and a base plate having a porous micro-nano structured surface with 1818 hydrophilic patterns was prepared according to the following procedure.
The experimental procedure was as follows:
1) Polishing the surface of the aluminum plate by sand paper to remove the oxide film;
2) The aluminum plate is treated by hot water bath, the hot water bath treatment condition is selected to be 75 ℃, and the time is 10min;
3) Thoroughly flushing with deionized water and blow-drying with nitrogen;
4) Treating the soleplate with oxygen plasma for 2 minutes;
5) Chemical vapor deposition: the plate was covered reversely with a petri dish and 10 μl of 1h,2 h-perfluorodecyl triethoxysilane was dropped onto the edge, and the plate was heated to 200 degrees for 30 minutes;
6) A photomask shielding base plate having a square void cell with a side length of 200 μm and a gap between each cell of 200 μm was used, and an ultraviolet ozone cleaner (185 nm,254nm,30mW/cm was used 2 ) And irradiating for 20min, and decomposing the hydrophobic film in the specific area through the photomask pattern to form 1818 hydrophilic patterns.
Example 5A substrate having a porous micro-nanostructure surface with 1620 hydrophilic patterns
An aluminum plate (purity: 99.5%, size: 63.5 mm. Times.63.5 mm, 1mm thick) was selected as a base plate material, and a base plate having a porous micro-nano structured surface with 1620 hydrophilic patterns was prepared as follows.
The experimental procedure was as follows:
1) Polishing the surface of the aluminum plate by sand paper to remove the oxide film;
2) The aluminum plate is treated by hot water bath, the hot water bath treatment condition is selected to be 75 ℃, and the time is 10min;
3) Thoroughly flushing with deionized water and blow-drying with nitrogen;
4) Treating the soleplate with oxygen plasma for 2 minutes;
5) Chemical vapor deposition: the plate was covered reversely with a petri dish and 10 μl of 1h,2 h-perfluorodecyl triethoxysilane was dropped onto the edge, and the plate was heated to 200 degrees for 30 minutes;
6) Using a metal sheet with a side length of 250 mum square vacant cells, a photomask with a gap of 200 μm between each cell was used to shield the substrate with an ultraviolet ozone cleaner (185 nm,254nm,30 mW/cm) 2 ) Irradiating for 20min, and decomposing the hydrophobic film in the specific area through the photomask pattern to form 1620 hydrophilic patterns.
Example 6A digital microfluidic chip with 1818 hydrophilic patterns
The transparent conductive ITO coated glass is used as a driving top plate, electrodes with the gap of 2.5 multiplied by 2.5mm and 60 mu m are formed by carving of a laser cutting machine, 18 electrodes are arranged in each channel, 6 channels are all arranged, and the electrodes of the six channels are sequentially connected in series to form a driving circuit for driving liquid drops of the 6 channels simultaneously. A 5 μm thick NOA68 insulating layer and a 100nm thick teflon hydrophobic layer were deposited on the drive top plate to prevent sample adhesion. The bottom plate prepared in example 4 was placed on the bottom layer, 400 μm thick glass was cut with a laser cutter into gaskets around 6 channels, placed in the middle, with the top plate electrode side facing inward, and placed on the top, to assemble a digital microfluidic chip. The preparation process and the structure schematic diagram are shown in fig. 3, the cross-sectional view of the DMF chip is shown in a in fig. 4, and 1818 hydrophilic patterns are shown in B in fig. 4. The prepared digital microfluidic chip is stored in an environment with the relative humidity of more than 60% before an experiment so as to ensure that water molecules are attached to the surface, and better droplet separation is realized.
Example 7A digital microfluidic chip with 2070 hydrophilic patterns
The preparation method was the same as in example 6, except that the base plate used was the base plate prepared in example 3.
Example 8A digital microfluidic chip with 1620 hydrophilic patterns
The preparation method was the same as in example 6, except that the base plate used was the base plate prepared in example 5. Effect example 1 Effect of superhydrophilic Pattern size on uniformity of generated droplets
To further achieve a uniform array of droplets, the present effect example tested the effect of the size of the superhydrophilic pattern on droplet uniformity.
To further achieve a uniform array of droplets, 3.5 μl of master containing the fluorescent Dye Reference Dye-FAM was instilled into each of the 6 channels of the different super hydrophilic pattern size DMF chips of examples 6-8. The corresponding electrodes in each channel are connected together and all droplets move together when a drive voltage is applied. In fig. 4C is shown a 3D surface map of droplets on different sized superhydrophilic patterns (150 μm, 200 μm and 250 μm) based on the fluorescence intensity observed using a fluorescence microscope. As observed, successful isolation of the tiny droplets onto the superhydrophilic pattern can be achieved, and no contamination from the superhydrophobic region is observed. Here, droplet height is used to further characterize droplet uniformity. The micro-droplet height is obtained by comparing the optical path difference between the reference droplet (parent droplet with a height of 400 μm) and the droplet (unknown height), and the height ratio is the ratio of the optical path (fluorescence intensity) under the fluorescence microscope. As shown in FIG. 4D, the heights of the droplets separated from the 150 μm, 200 μm and 250 μm superhydrophilic patterns were 43.20.+ -. 2.50 μm, 53.69.+ -. 1.56 μm and 74.36.+ -. 1.73 μm, respectively, and the corresponding volume changes were 7.1%, 3.4% and 2.9%. The effect on dNAA was negligible with less than 10% variability in partition volumes reported in the literature (Jim F Huggett, simon Cowen, carole A Foy, considerations for Digital PCR as an Accurate Molecular Diagnostic Tool, clinical Chemistry, volume 61,Issue 1,1January 2015,Pages 79-88). We subsequently selected a 200 μm pattern (example 6) to balance the uniformity and drop isolation of the subsequent dlap experiment.
Effect example 2 reaction volume accuracy test of digital microfluidic chip
The template concentration in each digital microfluidic chip is calculated according to poisson statistical principle as formula (1),
where λ is the average nucleic acid copy number per partition, k represents the number of positive partitions, and n represents the total number of partitions. The total number of nucleic acid molecules can be obtained by multiplying λ by n.
This equation provides the amount of nucleic acid, but obtaining the concentration requires knowledge of the exact reaction volume. In the DMF chip of example 4, 1080. Mu.L of n-hexadecane was first added to fill the chip (180. Mu.L of each channel, 6 channels total), and then the aqueous phase detection sample was added, and the droplet separation process effectively avoided evaporation and cross contamination due to the driving of the aqueous phase droplets in the oil phase. In the valve and the pipeline of the traditional pipeline microfluidic, liquid drops are easy to block and form dead volumes (the volumes are inaccurate due to liquid loss), the pipeline and the valve are not arranged in a DMF structure, the liquid drops are controlled by the electrode to freely move between the two plates, and any dead volumes possibly causing analysis errors are avoided. In this system, the exact reaction volume is equal to the difference in volume before and after separation (A in FIG. 5). To evaluate the unknown volume of the remaining parent droplets, the present invention measures the area occupied by a series of standard volume droplets and compares them to the unknown volume droplets.
First, droplets having a standard volume range of 1 to 6. Mu.L containing the fluorescent Dye Reference Dye-FAM were injected into DMF chips having a gap height of 400 μm (example 4) using a pipette, and a top-view fluorescence image of these droplets was obtained (B in FIG. 5). The normalized droplet area to volume relationship was then obtained using ImageJ. After fitting, assuming that the droplets in the sandwich DMF are oblate cylinders with a fixed height, there is a highly linear relationship between the droplet volume and its area (R 2 =0.999, C in fig. 5). As shown in FIGS. 5D and E, 5. Mu.L of a master droplet containing the fluorescent Dye Reference Dye-FAM was passed through 6 channels in sequence, and then fluorescent images were recorded. After calculation of the corresponding volume by comparing the area obtained with the standard curve of fig. 5C, a total of 2.12 μl of reagent was consumed by 1818 droplets in the 6 channels finally obtained. F in fig. 5 shows that the volumes consumed by each channel are 348.9nL, 349.5nL, 355.8nL, 352.2nL, 355.6nL, and 356.7nL, respectively. This corresponds to the volume of a single droplet: 1.15nL, 1.17nL, 1.16nL, 1.17nL, and 1.18nL (assuming that all droplets have the same volume). The volume change of each channel was less than 2.2%, further demonstrating the excellent uniformity of the superhydrophilic droplets. Highly uniform droplets and precise reaction volumes are DMF-basedDpap of the platform provides strong support.
Effect example 3 effect test of digital microfluidic detection System on nucleic acid quantification
The digital microfluidic detection system of this embodiment is shown in fig. 6, and includes a digital microfluidic chip (embodiment 7), a fluorescence microscope, DMF electronics, processing software, a temperature control board, a temperature sensor, a thermistor, and a power supply.
The schematic diagram of the digital microfluidic detection system for separating micro droplets and performing digital LAMP reaction in this embodiment is shown in FIG. 7.
In this example, nucleic acid was quantified by LAMP, and 25. Mu.l of the reaction system was shown in Table 1.
TABLE 1LAMP reaction System
The primer sequences (synthesized by biotechnology) are as follows:
F3：5’-GAATGCCCGTTCTGCGAG-3’(SEQ ID NO.2)。
B3：5’-TTCAGTTCCTGTGCGTCG-3’(SEQ ID NO.3)。
LF：5’-GGCGGCAGAGTCATAAAGCA-3’(SEQ ID NO.4)。
LB：5’-GGCAGATCTCCAGCCAGGAACTA-3’(SEQ ID NO.5)。
FIP：5’-CAGCATCCCTTTTCGGCATACCAGGTGGCAAGGGTAATGAGG-3’(SEQ ID NO.6)。
BIP：5’-GGAGGGTTGAAGAACTGCGGCAGTCGATGGCGTTCGTACTC-3’(SEQ ID NO.7)。
first, a series of lambda DNA dilutions (1 copy/. Mu.L, 10 copies/. Mu.L, 100 copies/. Mu.L, 1000 copies/. Mu.L) were prepared at a concentration ranging from 1 to 1000 copies/. Mu.L. According to the reaction system of this example, the LAMP mixture was simultaneously distributed along the prepared six-channel microarray. The Peltier heating source (temperature control plate) was directly located on the bottom surface of the digital microfluidic chip (example 4), and the bottom plate temperature of the LAMP reaction was stabilized at 65℃when a DC power of 8.6V, 1.7A was applied. dLAMP takes a fluorescent photograph at 65℃to measure the number of positive spots.
FIG. 8 shows the LAMP results (acquired every 10 minutes) on a real-time digital microfluidic detection system, indicating that the reaction was completed within 60 minutes.
As shown in fig. 9 a, an increase in lambda DNA concentration corresponds to an increase in the number of positive (fluorescent) spots. For samples ranging in concentration from 1 to 1000 copies/microliter, fluorescence image analysis was performed using ImageJ, obtaining 3, 30, 210, 1271 positive spots. From this, the corresponding DNA copy numbers were calculated to be 1.4 copies/microliter, 14 copies/microliter, 105 copies/microliter, and 1030 copies/microliter, respectively, using poisson statistics. As shown in FIG. 9B, the expected target concentration (measured by dsDNA HS assay fluorometer) of 1 to 1000 copies/microliter shows a linear relationship with the actual chip measured concentration (R 2 ＝0.9998)。
For comparison with fluorescence-based real-time quantitative LAMP (qLAMP), we also performed on-chip (non-DMF) relative quantitative experiments on different concentrations of lambda DNA (1 copy/. Mu.L, 10 copies/. Mu.L, 100 copies/. Mu.L, 1000 copies/. Mu.L, 10000 copies/. Mu.L). LAMP mixture was reacted at 65℃for 60 minutes using a commercial PCR apparatus (Bio-Rad, USA) with DNA templates of 1 copy/. Mu.L, 10 copies/. Mu.L, 100 copies/. Mu.L, 1000 copies/. Mu.L, 10000 copies/. Mu.L standards,
as shown in fig. 10 a, higher lambda DNA concentrations resulted in smaller cycle threshold (Ct). In FIG. 10B, a standard curve (R) is obtained by plotting Ct values from standard quantities 2 =0.989). The lambda DNA of unknown concentration was detected using the standard curve of dLAMP and the standard curve of qLAMP. As shown in fig. 9C, the quantitative results of the on-chip dwap (DMF-dwap) measurement and the quantitative results of the off-chip qLAMP (PCR instrument-qLAMP) measurement show good agreement. Furthermore, DMF-dlap showed less variation than commercial PCR instrument-qLAMP in three replicates. These results demonstrate the feasibility of rapid absolute quantification of lambda DNA with low detection limits and high accuracy based on dlap detection by the proposed digital microfluidic detection system.

Claims (10)

1. A backplane comprising a hydrophilic porous micro-nanostructure surface and a hydrophobic film;
preferably, the hydrophobic film does not completely cover the hydrophilic porous micro-nanostructure surface;
preferably, a hydrophilic pattern is formed on the surface of the hydrophilic porous micro-nano structure at a place which is not covered by the hydrophobic film;
preferably, the hydrophilic pattern is composed of a plurality of circular or rectangular unit cell arrays;
preferably, the individual diameter or side length of the unit cell is 50-500 μm;
preferably, the diameter or side length of the unit body is 150-250 μm;
preferably, the gap between the unit body and the adjacent unit body is 50-500 μm;
preferably, the gap between the unit body and the adjacent unit body is 150-250 μm.
2. The base plate of claim 1, wherein:
the bottom plate is made of one of beryllium, magnesium, aluminum, scandium, titanium, vanadium, chromium, iron, manganese, cobalt, nickel, copper, zinc, molybdenum, cadmium, indium, tin, antimony, bismuth, tantalum, thallium, lead, neodymium and erbium or an alloy consisting of the same;
preferably, the material of the bottom plate comprises one of magnesium, aluminum, zinc, iron, lead and manganese or an alloy consisting of the magnesium, the aluminum, the zinc, the iron, the lead and the manganese;
preferably, the material of the bottom plate comprises one of aluminum and magnesium or an alloy consisting of the aluminum and the magnesium;
preferably, the bottom plate is made of aluminum.
3. The base plate of claim 1, wherein:
the preparation raw material of the hydrophobic film is a hydrophobic material;
preferably, the hydrophobic material is an organosilane;
preferably, the organosilane comprises at least one of chlorotrimethylsilane, monochloro-propyl dimethyl silane, octyltrimethoxysilane, dodecafluoroheptyl propyl trimethoxysilane, tridecafluorooctyl triethoxysilane, trichloro (1 h,2 h-perfluorooctyl) silane, perfluorodecyl triethoxysilane, heptadecafluorodecyl trimethoxysilane, and heptadecafluorodecyl triethoxysilane;
preferably, the organosilane is perfluorodecyl triethoxysilane;
preferably, the hydrophobic film is a monolayer.
4. A method of manufacturing the base plate of any one of claims 1 to 3, comprising the steps of:
1) Forming a hydrophilic porous micro-nano structure surface on the bottom plate by adopting a water bath method;
2) Depositing a hydrophobic film on the bottom plate by adopting a vapor deposition method;
3) Ultraviolet rays are adopted to decompose the hydrophobic film in a specific area, so that a hydrophilic pattern is formed.
5. The method of manufacturing according to claim 4, wherein:
the step 1) of forming the hydrophilic porous micro-nano structure surface on the bottom plate by adopting a water bath method comprises the following specific steps: placing the bottom plate in a hot water bath for treatment;
preferably, the hot water bath treatment time is 0-24h;
preferably, the hot water bath treatment time is 2-60min;
preferably, the hot water bath treatment time is 5-20min;
preferably, the hot water bath treatment temperature is 50-100 ℃;
preferably, the hot water bath treatment temperature is 60-90 ℃;
preferably, the hot water bath treatment temperature is 70-80 ℃.
6. The method of manufacturing according to claim 4, wherein:
the vapor deposition method in the step 2) is a chemical vapor deposition method, and specifically comprises the following steps: depositing a hydrophobic material on the surface of the porous micro-nano structure to form a hydrophobic film;
preferably, the time of deposition is 10-60min;
preferably, the time of deposition is 25-35min;
preferably, the temperature of the deposition is 150-250 ℃;
preferably, the temperature of the deposition is 180-220 ℃.
7. The method of manufacturing according to claim 4, wherein:
in the step 3), ultraviolet rays are adopted to decompose the hydrophobic film in a specific area to form a hydrophilic pattern, and the method specifically comprises the following steps: using a photomask with a specific pattern to shield the bottom plate, and decomposing a hydrophobic film in a specific area through the photomask pattern by ultraviolet rays to form a hydrophilic pattern;
preferably, the ultraviolet rays are dual-wavelength ultraviolet rays;
preferably, the wavelength of the ultraviolet rays is 180-190nm and 250-260nm;
preferably, the ultraviolet irradiation time is 15-60min;
preferably, the photomask has a unit body through which ultraviolet rays can pass;
preferably, the unit body is circular or rectangular with a diameter or side length of 50-500 μm;
preferably, the diameter or side length of the unit body is 150-250 μm;
preferably, the gap between the unit body and the adjacent unit body is 50-500 μm;
preferably, the gap between the unit body and the adjacent unit body is 150-250 μm.
8. A microfluidic chip comprising a middle spacer, a drive top plate, and the bottom plate of claim 1;
preferably, the bottom plate is positioned at the bottom, the middle gasket is positioned in the middle, and the driving top plate is positioned above to form a three-layer structure;
preferably, the material of the middle gasket comprises at least one of glass, ceramic, metal and dimethyl siloxane;
preferably, the height of the middle gasket is 200-500 μm;
preferably, the driving top plate is a glass plate covered with a transparent conductive layer;
preferably, the thickness of the transparent conductive layer is 30-300nm;
preferably, the transparent conductive layer includes at least one of indium tin oxide ITO, aluminum doped zinc oxide AZO, and fluorine doped tin oxide FTO;
preferably, a driving circuit is arranged on one surface of the driving top plate facing the bottom plate;
preferably, the driving circuit forms 4-8 channels;
preferably, there are 12-24 electrodes per channel;
preferably, the electrode area comprises (2.2-2.8 mm) × (2.2-2.8 mm);
preferably, the electrode adjacent gap comprises 50-70 μm;
preferably, the side of the driving top plate facing the bottom plate is provided with an insulating coating;
preferably, the insulating coating surface has a hydrophobic coating;
preferably, the insulating coating comprises at least one of parylene or ultraviolet curing glue;
preferably, the ultraviolet curing glue comprises resin, photoinitiator and auxiliary agent;
preferably, the resin comprises at least one of phenolic resin, ABS resin, polyvinyl chloride resin, rosin, and amber;
preferably, the photoinitiator comprises at least one of benzoin ethers, phthalide ketals, acetophenones, benzophenones, thioarrowheads, aromatic sulfonium salts, iodonium salts, and ferrocenium salts;
preferably, the auxiliary agent comprises at least one of an industrially acceptable solvent, a stabilizer, a leveling agent, a defoaming agent, a plasticizer and a coupling agent;
preferably, the hydrophobic coating comprises teflon.
9. A detection method, wherein an oil phase liquid is added into the microfluidic chip according to claim 8, and then a detection sample is added for driving separation and detection;
preferably, the oil phase liquid comprises at least one of n-hexadecane, silicone oil and mineral oil;
preferably, the method is used for quantitative detection of nucleic acids;
preferably, the nucleic acid quantitative detection is applicable to at least one of a genetic mutation detection virus detection, a copy number variation detection, and a transgenic food detection;
preferably, the test sample comprises deoxyribonucleic acid and ribonucleic acid;
preferably, the test sample is lambda DNA.
10. A detection system comprising the microfluidic chip of claim 8, the system further comprising any one or more of:
1) A temperature control system;
2) A sample injection and discharge system;
3) A data processing system;
4) And a result display system.
CN202311645951.XA 2023-12-01 2023-12-01 Method and apparatus for DMF-based digital nucleic acid amplification systems Pending CN117866751A (en)

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